Abstract P040: Mst1 Physically Interacts with and Phosphorylates Beclin1, Thereby Negatively Regulating Autophagy in the Heart

Abstract

Autophagy mediates protein degradation and participates in protein quality control (QC). Inhibition of autophagy below physiological levels induces protein aggregation and cellular dysfunction. Beclin1 is a BH3-only protein that binds to Bcl-2. Bcl-2 negatively regulates autophagy through interaction with Beclin1. Here we show that the interaction between Bcl-2 and Beclin1 is regulated by mammalian STE 20 like-kinase-1 (Mst1), a proapoptotic kinase, in the heart. In Mst1 transgenic mice (Tg-Mst1), a model of dilated cardiomyopathy, accumulation of aggresomes was significantly increased and p62/SQSTM1, a protein degraded by autophagy, accumulated markedly in the myocardium. Autophagosome formation, as evaluated by GFP-LC3 dots, was suppressed in Tg-Mst1. The activity of Mst1 was suppressed by amino acid deprivation (AD) in cultured cardiomyocytes (CMs), and restoration of Mst1 activity attenuated AD-induced autophagy, as evaluated by LC3-II/ LC3-I and accumulation of p62/SQSTM1. Mst1 inhibited the kinase activity of the Beclin1-Vps34 (Class III PI3K) complex in CMs. ABT-737, a BH3 mimetic compound, reversed the Mst1-induced suppression of autophagy. Mst1-induced suppression of autophagy was alleviated when Bcl-2 were downregulated in CMs. Mst1 physically interacts with Beclin1 and stimulation of Mst1 enhances the interaction between Beclin1 and Bcl-2 in CMs. In vitro kinase assays using recombinant Beclin1 as substrate showed that Mst1 phosphorylates Beclin1. Using mass spectroscopy, we found that Mst1 phosphorylates Beclin1 at Thr 108 that located in its BH3 domain. Transduction of the Beclin1 Thr108Asp mutant adenovirus (Ad-T108D) attenuated AD-induced autophagy in CMs, and injection of Beclin1 Thr108Ala mutant adenovirus (Ad-T108A), but not Ad-T108D, restored starvation-induced LV dysfunction (LVEF: Ad-LacZ=0.51±0.05, Ad-T108D=0.53±0.04, Ad-T108A=.66±0.05, p<0.05) in beclin1 −/+ mice. Collectively, these results suggest that Mst1 inhibits autophagy through phosphorylation of Beclin1, enhancement of Beclin1-Bcl-2 interaction, and suppression of Vps34. Activation of Mst1 by stress suppresses autophagy below physiological levels and inhibits protein QC, which in turn may contribute to cardiac dysfunction.